Identification and Profiling of a Novel Bombyx mori latent virus Variant Acutely Infecting Helicoverpa armigera and Trichoplusia ni.

Insect cell expression systems are increasingly being used in the medical industry to develop vaccines against diseases such as COVID-19. However, viral infections are common in these systems, making it necessary to thoroughly characterize the viruses present. One such virus is Bombyx mori latent virus (BmLV), which is known to be specific to Bombyx mori and to have low pathogenicity. However, there has been little research on the tropism and virulence of BmLV. In this study, we examined the genomic diversity of BmLV and identified a variant that persistently infects Trichoplusia ni-derived High Five cells. We also assessed the pathogenicity of this variant and its effects on host responses using both in vivo and in vitro systems. Our results showed that this BmLV variant causes acute infections with strong cytopathic effects in both systems. Furthermore, we characterized the RNAi-based immune response in the T. ni cell line and in Helicoverpa armigera animals by assessing the regulation of RNAi-related genes and profiling the generated viral small RNAs. Overall, our findings shed light on the prevalence and infectious properties of BmLV. We also discuss the potential impact of virus genomic diversity on experimental outcomes, which can help interpret past and future research results.

Production of antiviral vaccine antigens using a silkworm-baculovirus expression system.

The outbreak of the SARS-2-CoV infection has become a global outbreak and continues to cause many deaths. In addition, the risk of pandemics continues to increase due to environmental changes and the globalization of human exchange and logistics. On the other hand, our preparedness for emerging infectious diseases caused by such unknown viruses is inadequate, and dealing with viral infections is one of the most important issues that need to be addressed immediately. Vaccine based disease control is considered an ideal countermeasure for infectious diseases, as it is expected to provide maximum efficacy at minimum cost. Although new nucleic acid-based vaccines are leading the way in the prevention of COVID-19, the mainstream of vaccines is still inactivated or live attenuated vaccines that use the pathogen virus itself. Subunit vaccines, in which specific virus-derived proteins are produced as recombinant proteins and used as vaccine antigens, have been developed, but production and development of many antigens that are difficult to mass-produce as recombinant proteins, such as the spike protein antigen of COVID-19 has not progressed. This paper describes the development of recombinant protein vaccines using the silkworm, which has an advantage in the production of such difficult-to-express vaccine antigens, especially virus-like particles.

Optimization of SARS-CoV-2 Spike Protein Expression in the Silkworm and Induction of Efficient Protective Immunity by Inoculation With Alum Adjuvants.

The newly emerged severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing a spread of coronavirus disease 2019 (COVID-19) globally. In order to end the COVID-19 pandemic, an effective vaccine against SARS-CoV-2 must be produced at low cost and disseminated worldwide. The spike (S) protein of coronaviruses plays a pivotal role in the infection to host cells. Therefore, targeting the S protein is one of the most rational approaches in developing vaccines and therapeutic agents. In this study, we optimized the expression of secreted trimerized S protein of SARS-CoV-2 using a silkworm-baculovirus expression vector system and evaluated its immunogenicity in mice. The results showed that the S protein forming the trimeric structure was the most stable when the chicken cartilage matrix protein was used as the trimeric motif and could be purified in large amounts from the serum of silkworm larvae. The purified S protein efficiently induced antigen-specific antibodies in mouse serum without adjuvant, but its ability to induce neutralizing antibodies was low. After examining several adjuvants, the use of Alum adjuvant was the most effective in inducing strong neutralizing antibody induction. We also examined the adjuvant effect of paramylon from Euglena gracilis when administered with the S protein. Our results highlight the effectiveness and suitable construct design of the S protein produced in silkworms for the subunit vaccine development against SARS-CoV-2.

[Production of antiviral vaccines using a silkworm-baculovirus expression system].

The outbreak of the new coronavirus (SARS-2) infection (COVID-19) in Wuhan, China, has become a global outbreak and continues to cause many deaths. Not only COVID-19, but the risk of pandemic continues to increase due to global environmental changes and globalization of human exchange and logistics. On the other hand, we are inadequately prepared for such unknown emerging infectious diseases, and dealing with viral infections has become one of the most important issues facing humanity. Vaccine based disease prevention is considered an ideal disease control strategy, especially for viral diseases for which there is no or little cure. New nucleic acid vaccines (mRNA and viral vector vaccines) are leading the way in the fight against COVID-19, but even today, the mainstream of vaccines are inactivated vaccines and live-attenuated vaccines. Subunit vaccines, in which some specific viral proteins of the pathogen are produced as recombinant proteins and used as vaccine antigens, have also been developed. On the other hand, many antigens that are difficult to produce in large quantities as recombinant proteins, such as the spike protein antigen of COVID-19 (the same antigen is also targeted by nucleic acid vaccines), have not yet been produced and developed. In this paper, we describe the development of a recombinant protein vaccine based on the silkworm insect factory, which has an advantage in the production of such difficult-to-express vaccine antigens.

Production of scFv, Fab, and IgG of CR3022 Antibodies Against SARS-CoV-2 Using Silkworm-Baculovirus Expression System.

COVID-19, caused by SARS-CoV-2, is currently spreading around the world and causing many casualties. Antibodies against such emerging infectious diseases are one of the important tools for basic viral research and the development of diagnostic and therapeutic agents. CR3022 is a monoclonal antibody against the receptor binding domain (RBD) of the spike protein (S protein) of SARS-CoV found in SARS patients, but it was also shown to have strong affinity for that of SARS-CoV-2. In this study, we produced large amounts of three formats of CR3022 antibodies (scFv, Fab and IgG) with high purity using a silkworm-baculovirus expression vector system. Furthermore, SPR measurements showed that the affinity of those silkworm-produced IgG antibodies to S protein was almost the same as that produced in mammalian expression system. These results indicate that the silkworm-baculovirus expression system is an excellent expression system for emerging infectious diseases that require urgent demand for diagnostic agents and therapeutic agents.

Efficient production of recombinant SARS-CoV-2 spike protein using the baculovirus-silkworm system.

In the case of a new viral disease outbreak, an immediate development of virus detection kits and vaccines is required. For COVID-19, we established a rapid production procedure for SARS-CoV-2 spike protein (S protein) by using the baculovirus-silkworm expression system. The baculovirus vector-derived S proteins were successfully secreted to silkworm serum, whereas those formed insoluble structure in the larval fat body and the pupal cells. The ectodomain of S protein with the native sequence was cleaved by the host furin-protease, resulting in less recombinant protein production. The S protein modified in furin protease-target site was efficiently secreted to silkworm serum and was purified as oligomers, which showed immunoreactivity for anti-SARS-CoV-2 S2 antibody. By using the direct transfection of recombinant bacmid to silkworms, we achieved the efficient production of SARS-CoV-2 S protein as fetal bovine serum (FBS)-free system. The resultant purified S protein would be useful tools for the development of immunodetection kits, antigen for immunization for immunoglobulin production, and vaccines.